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Phenotypic Alteration and Target Gene Identification Using Combinatorial Libraries of Zinc Finger Proteins in Prokaryotic Cells

Kyung-Soon Park, Young-Soon Jang, Horim Lee, and Jin-Soo Kim^*

ToolGen, Inc., 461-6 Jeonmin-dong, Yuseong-gu, Daejeon, South Korea 305-390

Received 9 March 2005/ Accepted 27 April 2005

	ABSTRACT

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We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified ubiX, whose down-regulation correlates with the thermotolerance phenotype in Escherichia coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies.

	TEXT

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Engineered zinc finger proteins (ZFPs), either those by themselves or those that have been fused to transcriptional activation or inhibition domains, have been used to regulate the transcription of numerous target genes in eukaryotic cells (2, 4, 9, 10, 13). Recent advances in the field allow one to construct ZFP libraries that consist of tens of thousands of active transcription factors (3, 6, 7, 8). Such libraries have been used successfully to scan the yeast and mammalian genomes for genes that induce phenotypic alterations in cells when their expression is activated or inhibited (3, 6, 8).

ZFPs that have been linked to the alpha subunit of Escherichia coli RNA polymerase have been shown to bind to and activate transcription of a reporter gene in E. coli (5). However, it is not yet clear whether engineered ZFPs can regulate the expression of endogenous genes and induce phenotypic changes in prokaryotic cells.

Here we show that combinatorial libraries of ZFPs can be expressed and can induce phenotypic alterations in E. coli. Using this method, we identified a target gene whose repression by ZFP is responsible for thermotolerance in E. coli.

Creating phenotypic alterations with ZFP libraries in E. coli. We have constructed combinatorial libraries that encode ZFPs by using as modular building blocks well-characterized zinc fingers that display diverse DNA-binding specificities. Individual zinc fingers recognize three-base-pair subsites but cannot bind stably to target DNA. In general, at least three zinc fingers must be stitched together to constitute a functional DNA-binding ZFP. We combined DNA fragments that encode 40 different zinc fingers with distinct DNA-binding specificities and randomly shuffled them to generate a diverse collection of three-fingered and four-fingered ZFPs (8). Thus, the complexities of the resulting libraries are 6.4 x 10⁴ and 2.6 x 10⁶, respectively. This population of ZFP genes was cloned into an expression plasmid, pZL1, which was modified from pBT-LGF2 (Clontech) to have a V5 epitope and multiple cloning sites. The ZFP library was constructed by subcloning three-finger and/or four-finger ZFP inserts isolated from our yeast ZFP library into the EcoRI and NotI sites of pZL1 (8). No additional sequences that encode transcriptional effector domains were fused to the ZFP genes.

These ZFP libraries were expressed in E. coli DH5, and cells were screened for thermotolerance phenotype. The screens were conducted under conditions in which more than 99.9% of wild-type cells died.

We screened 10 million transformants, which exceeded the complexities of the libraries, for E. coli cells that became resistant to heat after transformation with the ZFP library. E. coli ZFP transformants were incubated at 50°C for 2 h before being plated on rich medium. Plasmids were purified from 23 individual colonies that survived the heat treatment. Ten different ZFPs were identified (Table 1), and the levels of thermotolerance of selected ZFP transformants were compared with that of control cells. Under the experimental conditions described above, less than 0.01% of the control cells survived. In sharp contrast, up to 6% of cells transformed with certain ZFPs survived these extreme conditions (Fig. 1A).

View larger version (42K): [in this window] [in a new window]   FIG. 1. Phenotypic changes in E. coli induced by artificial ZFPs. (A) Thermotolerant phenotype induced by ZFPs. Selected clones (T-1 to T-10) were cultured for 2 h at 37°C (no treatment) or 50°C (heat shock) in LB containing 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) and then plated on LB plates. C, E. coli cells transformed with control plasmid pZL1; T, E. coli cells transformed with thermotolerance-inducing ZFPs. (B) Site-directed mutagenesis of the T9 ZFP. The arginine residue in the QTHR1 zinc finger was mutated to alanine, and the ability of the resulting mutated ZFP (designated T9-M) to induce thermotolerance in E. coli was tested. The phenotypic changes were confirmed by plasmid rescue, sequence analysis, and retransformation of E. coli. The triangles drawn above each panel indicate 10-fold serial dilutions (1:1 to 1:10,000, left to right) of spotted cells.

View larger version (35K): [in this window] [in a new window]   FIG. 2. Identification of the target gene regulated by T9 that gave rise to the thermotolerance phenotype. (a) Effect of T9 expression on the concentration of ubiX RNA. For the analysis of UbiX gene expression, cDNA synthesis was performed on RNA with a UbiX-R primer (5'-CTG GAA AGA ACC GGA AGA GAT GCT G-3'). Real-time reverse transcription-PCR was performed by using a light cycler (Corbett Research) with the UbiX-F (5'-TGA AAC GAC TCA TTG TAG GCA TCA G-3') and UbiX-R primer set. The concentration of RNA encoding glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. C, E. coli cells transformed with pZL1; T9, T9 ZFP transformants. (b) Interaction of T9 with potential binding sites located in the ubiX promoter. ubiX, ubiX knockout mutant; T9, T9 ZFP transformants; Ab, antibody. (c) Disruption of the ubiX gene in E. coli and analysis of thermotolerance. E. coli strain DY330 [W3110 DlacU169 gal490 lcI857 D (cro-bioA)] was used for gene disruption by homologous recombination. Gene knockout was performed by targeted homologous recombination as described previously (12). The position of a potential T9-binding site relative to the translation start site is indicated. Binding of the T9 ZFP to this site was confirmed by immunoprecipitation. In contrast, the T9-M protein was unable to bind to this site. In panel c, the triangles drawn above each panel indicate 10-fold serial dilutions (1:1 to 1:10,000, left to right) of spotted cells. ubiX, ubiX knockout mutant; T9, T9 ZFP transformants; C, E. coli cells transformed with pZL1.

In order to investigate whether the transcription of any of the open reading frames (ORFs) adjacent to the T9 binding sites in the six clones was actually regulated by T9, we subjected each of the ORFs to real-time reverse transcription-PCR. Among the six ORFs tested, we found that transcription of the ORF that encodes UbiX, which is involved in the biosynthesis of ubiquinone, was decreased more than twofold upon expression of the T9 ZFP (Fig. 2a). The transcript levels of the other five clones were not affected by the expression of T9. Because the T9 ZFP was not fused to an effector domain, T9 is expected to function as a transcriptional repressor in E. coli. A T9 binding site with a one-base mismatch was identified at 90 base pairs upstream of the ubiX start codon. The in vivo binding of T9 to the putative target site upstream of the ubiX gene was confirmed by the immunoprecipitation procedure described above followed by PCR (Fig. 2b). As expected, the mutant T9-M protein was not able to bind to this site in vivo (Fig. 2b) or to down-regulate the transcription of the ubiX gene (data not shown).

To validate the functional relevance of ubiX to the thermotolerance phenotype in E. coli, we knocked out the ubiX gene and examined the response of the mutant strain to heat treatment. The ubiX mutant strain grew slowly compared to the wild-type strain, and its colony size on the culture plate was relatively small under normal growth conditions. However, the ubiX mutant strain was extremely resistant to the lethal effects of heat shock (Fig. 2c). In contrast to observations with the ubiX mutant strain, no defect in growth was detected in the T9-expressing strain. This result suggests that a regulation of target gene expression that is more moderate than can be achieved by gene knockout could be important in the generation of certain bacterial phenotypes that would be useful for microbial production of industrial products.

In this study, we demonstrated that ZFPs can function as efficient transcription factors in E. coli. In addition, ZFPs provide novel tools for determining which genotypes give rise to specific phenotypes.

There are several advantages of using ZFP transcription factors as genome-wide regulators of gene expression in microorganisms. First, ZFPs can cause subtle changes in gene expression. In cases in which overexpression or knockout of a gene is harmful to the organism, subtle changes in gene expression might allow one to achieve the desired phenotypes. Second, phenotypic transfer from one strain to another strain of the same species is straightforward with our approach. This would be of particular importance for certain industrial strains of bacteria that are difficult to manipulate genetically. Third, it should be possible to conditionally produce desired phenotypes by using an inducible promoter to express a ZFP. Our results therefore suggest that the ZFP library approach is a useful tool for engineering and studying prokaryotic microorganisms.

	ACKNOWLEDGMENTS

 
We thank W. Seol, H. C. Shin, and J. W. Park for helpful discussions and K. LaMarco for carefully reading our manuscript.

This work was supported by the National Research Laboratory Program (grant M1-0104-00-0048) from the Korean Ministry of Science and Technology.

	FOOTNOTES

 
* Corresponding author. Mailing address: ToolGen, Inc., 461-6 Jeonmin-dong, Yuseong-gu, Daejeon, South Korea 305-390. Phone: 82-42-863-8166. Fax: 82-42-863-3840. E-mail: jsk{at}toolgen.com.

These authors contributed equally to this work.

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